Innate compensatory responses after ischemic injury can attenuate the extent of ischemic injury and promote functional recovery. Exogenous delivery of angiogenic factors has been shown to enhance this innate response. This research project is aimed primarily at overcoming certain barriers to the translation of the "enhanced innate response to injury" paradigm to the clinical setting. For example, systemic delivery of therapeutic proteins and genes into the brain is prevented by the brain blood barrier (BBB), and intraventricular delivery results in non-specific angiogenic factor distribution and gene expression, which can cause unwanted angiogenesis in normal tissues and untoward side effects. Moreover, stereotactic injection of proteins and vectors into the ischemic penumbra requires an invasive procedure and can cause additional damage. We have generated a custom-designed adeno-associated viral vector (AAV) with two primary attributes that suit our intended purpose. First, the vector's hypoxia response elements (HRE) restricts transgene expression to ischemic tissue. Second, AAV serotype nine (AAV9) effectively pentrates the BBB, enabling intravenous adminsitration. We have named our vector H9. We propose to use intravenous (IV) delivery of H9 vectors to achieve targeted gene expression in brain ischemic foci. The payload of the novel vector will be both vascular endothelial growth (VEGF) and angiopoietin-1 (Ang-1). VEGF was chosen, because it is the most- studied angiogenic factor for ischemic stroke therapy, has angiogenic and neurogenic effects, and promotes both neuronal protection and restoration. Ang-1 was chosen, because it reduces vessel leakage caused by VEGF and works synergistically with VEGF on angiogenesis and neuroprotection. The goal of this project is to develop an innovative approach to improve the outcomes of gene-based therapies for ischemic brain injury. We plan to use a mouse permanent distal middle cerebral artery occlusion model to test our hypotheses, that (1) IV injection of H9 vector will result in targeted gene expression in the ischemic brain and (2) IV injection of H9-VEGF and H9-Ang-1 results in better outcomes than stereotactic injection. This pilot study will provide solid proof of principle for our approach, and in subsequent applications (R01), we will extend the studies to optimize vector delivery (dosage and delivery schemes);to better understand the mechanisms of the neuroprotective or neurorestorative effects of VEGF and Ang-1, and neurobehavioral recovery;and to study the feasibility of this approach for clinical application. The proposed technology is novel and, if successful, will lead to development of a more selective gene therapy for ischemic stroke. The same approach can also be applied to the treatment of spinal cord injury. PUBLIC HEALTH RELEVANCE: This project will develop a new method to induce new functional small blood vessels in the brain where the blood supply is insufficient. As result, increased blood flow will protect neurons from death.